PROJECT SUMMARY/ABSTRACT Adoptive cell therapy (ACT) with tumor-reactive T cells isolated from the blood or tumor, or engineered with T cell receptors (TCRs) or chimeric antigen receptors (CARs) by gene transfer, is providing new treatment options for patients with chemotherapy refractory malignancies. Developing effective ACT for common cancers such as breast and lung cancer represents an urgent unmet need, but has posed several obstacles. These include the identification of molecules expressed on tumor cells that can be targeted safely and the design of optimal CARs that both redirect T cells to recognize tumors and overcome the immunosuppressive local tumor microenvironment that interferes with T cell mediated tumor eradication. We have identified the receptor tyrosine kinase-like orphan receptor ROR1 as a promising target for CAR-T cell therapy of human malignancies, including refractory chronic lymphocytic leukemia, mantle cell lymphoma, triple negative breast cancer and non small cell lung cancer. We designed and optimized ROR1-specific CARs for recognition of ROR1 positive tumors in vitro and for efficacy in immunodeficient mice engrafted with human tumor xenografts, and demonstrated that transferring autologous ROR1 CAR-T cells in non-human primates was safe. We have developed a monoclonal antibody to identify patients that have ROR1 positive tumors and produced a clinical grade ROR1 CAR lentiviral vector. Thus, we are now poised to perform a first-in-human clinical trial targeting ROR1 in both hematologic malignancies and solid tumors and to develop next generation ROR1 CARs designed to improve safety and for superior function in the tumor microenvironment. The specific aims are: Aim 1: To determine the feasibility and safety of targeting ROR1 with autologous CD8+ and CD4+ T cells engineered with a ROR1/4-1BB/CD3? CAR and formulated in a defined composition for patients with hematologic malignancies (Cohort A) and solid tumors (Cohort B). Aim 2: To evaluate CAR-T cell phenotype, function and trafficking to tumor sites in vivo, and determine potential mechanisms of tumor resistance. Aim 3: To evaluate novel ROR1 CAR designs that enhance selective tumor cell recognition and/or overcome negative signaling by PDL1.